U.S. patent application number 14/784203 was filed with the patent office on 2016-03-17 for antioxidant extract from brown macroalgae and method for obtaining same.
The applicant listed for this patent is UNIVERSIDADE DE SANTIAGO DE COMPOSTELA. Invention is credited to Maria Jose NUNEZ GARCIA, Marivel SANCHEZ GUERRERO, Jorge SINEIRO TORRES.
Application Number | 20160074317 14/784203 |
Document ID | / |
Family ID | 50112562 |
Filed Date | 2016-03-17 |
United States Patent
Application |
20160074317 |
Kind Code |
A1 |
SINEIRO TORRES; Jorge ; et
al. |
March 17, 2016 |
ANTIOXIDANT EXTRACT FROM BROWN MACROALGAE AND METHOD FOR OBTAINING
SAME
Abstract
The invention relates to a method for obtaining antioxidant
extracts from macroalgae using ultrasound-assisted continuous
aqueous extraction. The process can be performed using fresh alga
or dry alga, after resuspending same in water. A suspension of alga
in water is prepared with a solid concentration of between 10 and
30%. The mixture is fed to an ultrasonic disruption system. The
extract is filtered and lyophilized, obtaining total polyphenol
concentrations of 62.4 mg eq. of phloroglucinol/g of lyophilisate
with Bifurcaria bifurcata and 44 mg eq. of phloroglucinol/g of
lyophilisate with Ascophyllum nodosum. The extract can be used as
an ingredient in cosmetic and food formulations.
Inventors: |
SINEIRO TORRES; Jorge;
(Santiago de Compostela, ES) ; SANCHEZ GUERRERO;
Marivel; (Santiago de Compostela, ES) ; NUNEZ GARCIA;
Maria Jose; (Santiago de Compostela, ES) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSIDADE DE SANTIAGO DE COMPOSTELA |
Santiago de Compostela |
|
ES |
|
|
Family ID: |
50112562 |
Appl. No.: |
14/784203 |
Filed: |
April 10, 2014 |
PCT Filed: |
April 10, 2014 |
PCT NO: |
PCT/ES2014/070288 |
371 Date: |
October 13, 2015 |
Current U.S.
Class: |
424/401 ;
424/195.17; 426/238; 426/541 |
Current CPC
Class: |
A61K 2800/522 20130101;
A61Q 19/00 20130101; A61K 8/9789 20170801; A61K 8/9711 20170801;
A61K 8/347 20130101; A61K 36/03 20130101; A23V 2200/00 20130101;
A23L 3/3463 20130101; A23L 17/60 20160801; A61K 2236/00 20130101;
A61K 2800/10 20130101; A23L 33/105 20160801; A61K 2800/84 20130101;
A61K 2800/82 20130101; A23V 2002/00 20130101; A23V 2002/00
20130101; A23V 2200/02 20130101; A23V 2250/202 20130101 |
International
Class: |
A61K 8/97 20060101
A61K008/97; A23L 3/3463 20060101 A23L003/3463; A23L 1/337 20060101
A23L001/337 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 12, 2013 |
ES |
P201330523 |
Claims
1. A composition comprising an aqueous antioxidant extract of at
least one species of brown macroalgae, a lyophilized derivative of
said aqueous antioxidant extract, or a mixture thereof, wherein:
said aqueous antioxidant extract being obtained by extraction of
tissue from at least one species of brown macroalgae, wherein said
tissue has been subjected to a cell disruption process by
continuously applying ultrasound with a power density of between 3
and 13 W/cm.sup.3; said aqueous antioxidant extract having: a) a
carbohydrate content between 33 and 156 mg of glucose/g of extract
once lyophilized; b) a fucoidan content, said fucoidans expressed
as total sulfates after digesting the sample, between 44 and 118 mg
of sulfates/g once lyophilized; c) a phlorotannin content, said
phlorotannins expressed as phloroglucinol, between 12 and 62.4 mg
equivalents of phloroglucinol/g once lyophilized; and d) an
alginate content between 10 and 55 mg equivalents of glucuronic
acid/g once lyophilized.
2. A composition according to claim 1, wherein said composition has
a maximum absorbance in the ultraviolet region between 260-280
nm.
3. A composition according to claim 1, wherein said composition has
an inhibitory capacity of DPPH radical with an EC.sub.50 value of
17.7 to 23.7 mg of extract/ml.
4. The extract according to claim 1, where the brown macroalga is
selected from the group consisting of Bifurcaria bifurcata,
Ascophyllum nodosum, Saccorhiza polyschide, Sargassum muticum, and
mixtures thereof.
5. A method for obtaining an antioxidant extract, from fresh or dry
brown macroalgae, which comprises: a) washing the macroalgae with
water if fresh macroalgae are used; b) mixing the macroalga with
water with a liquid/solid (L/S) ratio between 3 and 5 g/g; c)
grinding the mixture obtaining a ground macroalga with a particle
size less than 3 mm; d) mixing the ground macroalga with a liquid
solvent comprising pure ethanol or ethanol:water mixtures at a 1:1
v/v ratio, at a liquid/solid (L/S) ratio of between 5 and 15 (g/g)
when starting from fresh macroalga and between 50 and 150 (g/g)
when starting from dry macroalga; e) subjecting the mixture to a
cell disruption process by continuously applying ultrasound with a
power density in the range between 3 and 13 W/cm.sup.3; f)
separating the solid algal residue obtained in the preceding step
by means of sedimentation and subsequent centrifugation or
filtration; g) removing the ethanol or 50% of the water under
vacuum at a temperature of less than 40.degree. C., obtaining a
concentrated extract; and h) lyophilizing the concentrated extract
obtaining a lyophilized extract.
6. The method according to claim 5, wherein the ground macroalga
have a particle size of between 0.5 and 2 mm for dry macroalga and
between 0.5 and 3 mm for fresh macroalga.
7. (canceled)
8. A cosmetic formulation comprising a composition according to
claim 1.
9. A food comprising a composition according to claim 1.
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The present invention relates to the extraction of compounds
from macroalgae. More specifically, the invention relate to the
extraction of antioxidants from brown macroalgae.
STATE OF THE ART
[0002] Antioxidants are secondary metabolites present in
macroalgae, plants, and particularly fruits. They are compounds
that inhibit or prevent oxidation of a substrate. In the food,
pharmaceutical and cosmetics industry are used primarily synthetic
antioxidant compounds, such as butylated hydroxytoluene (BHT),
propyl gallate or butyrohydroxyanisole (BHA). Natural antioxidants
like those originating from grape extract, rosemary extract, cocoa
extract, etc., are being accepted better due to their low toxicity
and high antioxidant activity. A large family of antioxidant
compounds are phlorotannins present in macroalgae which show good
antioxidant activity.
[0003] Several protocols for the extraction of phlorotannins have
been proposed, among which it is possible to mention sequential
liquid-liquid partitions, using primarily organic solvents such as
methanol, hexane, dichloromethane, chloroform, ethyl acetate and
butanol, in addition to purifications in chromatographic columns,
such as Antioxidant Effects of Phlorotannins Isolated from Ishige
okamurae in Free Radical Mediated Oxidative Systems, Yanping, Z.,
Zhong-Ji Q., Yong L., Moon-Moo K., Sang-Hoon, L. & Se-Kwon K,
J. Agric. Food Chem., 56, 7001-7009, 2008; Phenolic Compounds in
the Brown Seaweed Ascophyllum nodosum: Distribution and
Radical-scavenging Activities, Audibert, L., Fauchon, M., Blanc,
N., Hauchard, D. & Ar Galla, E, Phytochemical analysis, 21,
399-405, 2010; Chemical components and its antioxidant properties
in vitro: an edible marine brown alga Ecklonia cava, Li, Y., Qian,
Z. J., Ryu, B., Lee, S. H., Kim, M. M. and Kim, S. K., Bioorg Med
Chem 17, 1963-1973, 2009; Distribution and radical scavenging
activity of phenols in Ascophyllum nodosum (Phaeophyceae), Breton,
F., Cerantola, S. & Ar Gall E., Journal of Experimental Marine
Biology and Ecology, 399, 167-172, 2011. However these processes
generate residues that are not allowed in food and cosmetics, such
as methanol or organohalogen compounds. Furthermore, if a polarity
that is lower than that of water is required, the hydroalcoholic
mixtures are an option as opposed to petroleum solvents.
[0004] The use of 70% acetone and ethyl acetate as solvents was
reported in Phlorotannins as Radical Scavengers from the Extract of
Sargassum ringgoldianum, Nakai, M., Kageyama, N., Nakahara, K.
& Miki, W., Marine Biotechnology, 8, 409-414, 2006). A first
lipid material removal phase with hexane and subsequent extraction
of phlorotannins with acetone-water at 70% was used in
High-performance liquid chromatographic analysis of phlorotannins
from the brown alga Fucus vesiculosus, Koivikko, R., Loponen, J.,
Pihlaja, K. and Jormalainen, V., Phytochem. Anal. 18, pp. 326-332,
2007. Pure ethanol and 60% ethanol have been used for extraction of
phlorotannins in species such as Ascophyllum nodosum and Fucus
vesiculosus, followed by liquid-liquid partition with petroleum
ether or dichloromethane (Evaluation of quantitative methods for
the determination of polyphenols in algal extracts, Parys, R.,
Rosenbaum, A., Kehraus, S., Reher, G., Glombitza, K-W and Konig, G.
M. J. Nat. Prod., 1865-1870, 2007.
DESCRIPTION OF THE INVENTION
[0005] The extracts obtained from processing brown macroalgae, such
as Bifurcaria bifurcata and Ascophyllum nodosum, can be employed
for food, cosmetic and/or pharmaceutical use due to their fucoidan
and phlorotannin content. The high fucoidan content leads to these
extracts having a wetting capability on skin, whereas the
antioxidant power is due to the presence of phlorotannins.
[0006] Obtaining a natural product that does not have a strong
characteristic aroma is desirable for use as a cosmetic ingredient
in order to not interfere with other desired uses, which is favored
by aqueous extraction and by not using organic solvents. Another
desirable feature of the process of extraction of extracts is for
the solvents that are used to be inexpensive, renewable, having
little or no toxicity and for the handling thereof to not be
dangerous. Crude extracts or isolated fractions often show greater
antioxidant activity than synthetic antioxidants such as BHA
(butyrohydroxyanisole) or BHT (butyrohydroxytoluene).
[0007] The method object of the present invention allows using
water to obtain a stable product that is easy to handle and add to
different products and free of trace solvents.
[0008] The method of extraction of the antioxidant from a brown
macroalga comprises:
[0009] a) If fresh macroalga is used, it is subjected to washing
with running water, removing sand and epiphytes, and is then dried
with absorbent paper. If dry alga is used, this process of cleaning
with water is not necessary.
[0010] b) The dry or fresh alga is then mixed with water at a
liquid/solid (L/S) ratio of between 3 and 5 g/g.
[0011] c) The mixture from the preceding step is then subjected to
a grinding process, preferably in a blade grinder, until reducing
the particle size between 0.5 and 2 mm for dry macroalga and
between 0.5 and 3 mm for fresh macroalga.
[0012] d) Mixing the ground macroalga with a liquid solvent
comprising pure ethanol or ethanol:water mixtures at a 1:1 v/v
ratio, at a liquid/solid (L/S) ratio of between 5 and 15 (g/g) when
starting from fresh macroalga and between 50 and 150 (g/g) when
starting from dry macroalga,
[0013] e) subjecting the mixture to a cell disruption process by
continuously applying ultrasound.
[0014] f) The solid algal residue obtained in the preceding step is
separated by sedimentation and subsequent centrifugation or
filtration.
[0015] g) The ethanol or 50% of the water, only if water had been
used, is removed under vacuum without exceeding the temperature of
40.degree. C.
[0016] h) The concentrated extract is lyophilized to obtain a solid
extract.
[0017] The product is stored cooled to less than 5.degree. C. and
protected from the light to prevent it from being altered.
[0018] In another aspect, the invention relates to a stable
antioxidant extract obtained from processing brown macroalgae, more
specifically macroalgae algae of the Bifurcaria bifurcata,
Ascophyllum nodosum, Saccorhiza polyschides and Sargassum muticum
species.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The embodiments shown in detail in the drawings are
illustrated by way of non-limiting example:
[0020] FIG. 1 shows the diagram for ultrasound-assisted cell
disruption in a continuous macroalgal feed system.
[0021] FIG. 2 shows the detection of phlorotannins by means of C18
reversed-phase column HPLC.
[0022] FIG. 3 shows the ultraviolet profile of the Bifurcaria
bifurcata extract, with a characteristic phlorotannin profile that
is similar to that of phloroglucinol.
EMBODIMENT OF THE INVENTION
[0023] In a particular embodiment the invention relates to an
aqueous antioxidant extract obtained from brown macroalgae
characterized by a) a carbohydrate content between 33 and 156 mg of
glucose/g of lyophilizate; b) a fucoidan content, said fucoidans
expressed as total sulfates after digesting the sample, between 44
and 118 mg of sulfates/g of lyophilizate; c) a phlorotannin
content, said phlorotannins expressed as phloroglucinol, between 12
and 62.4 mg equivalents of phloroglucinol/g of lyophilizate; and d)
an alginate content between 10 and 55 mg equivalents of glucuronic
acid/g of lyophilizate.
[0024] In another aspect, the invention relates to a cosmetic
and/or food composition comprising the antioxidant extract object
of the present invention.
[0025] An example of the application of the method of extraction of
antioxidants from Bifucaria Bifurcata is mentioned by way of
example and without limiting the scope of protection. The
extraction is performed continuously and in temperature conditions
less than 40.degree. C. to prevent a reduction in the antioxidant
power or the amount of the compounds that are extracted.
[0026] FIG. 1 shows the diagram for ultrasound-assisted cell
disruption in a continuous macroalgal feed system made up of an
ultrasound generating unit including a sonotrode (105) arranged in
a flow-through cell (104), which is fed by means of a main
peristaltic pump (102) and another recirculating pump (103).
[0027] In a particular embodiment of the invention the process of
extraction of the aqueous antioxidant compound comprises the
following steps:
[0028] a) 120 g of fresh macroalga were washed with water fit for
drinking and subsequently with distilled water.
[0029] b) 300 ml of water were added.
[0030] c) The macroalga was pre-ground by operating a blade mixer
for 10 min, obtaining a particle size of less than 3 mm.
[0031] d) Water was added to the preparation obtained in the
preceding step until obtaining an L/S ratio of 10, and it was
introduced in a feed tank (100) which is mixed by means of an
agitator (101). One of the extraction conditions described in Table
1, which summarizes a 2.sup.2 factorial experimental design with 4
center points, was selected.
[0032] e) The continuous ultrasonic extraction conditions
considered the parameters of power (as amplitude or percentage of
nominal power applied) and ratios between the recirculation of the
inflow rate of the algal broth into the flow-through cell
(sonication or ultrasonic cell) (104) in which a sonotrode (105)
continuously applies ultrasound to the mixture with a power density
in the range of between 3 and 13 W/cm.sup.3. Table 1 summarizes the
combination of treatments performed for the experimental design.
The extraction set-up requires an ultrasonic-assisted extraction
system such as the one shown in FIG. 1.
[0033] f) The sedimentable solid residue was left to settle and was
separated from the supernatant by decanting.
[0034] g) The next step consisted of centrifuging and separating
the supernatant again. The supernatant has a volume of 1200 ml,
which was reduced to 500 ml by vacuum evaporation at 30.degree. C.
or at room temperature,
[0035] h) A lyophilized extract (106) was obtained which, in this
particular embodiment, comprises a total volume of 1400 ml in which
and finally to obtain a solid and dry extract.
TABLE-US-00001 TABLE 1 4 center point, 2.sup.2 experimental design.
Amplitude adjustment codes -1, 0 and 1 correspond to amplitudes of
50%, 70% and 90%. Recirculation ratio codes -1, 0 and +1 correspond
to 1, 1.5 and 2, the recirculation flow rate being constant of 300
ml/min Residence Recirculation Power time in Amplitude ratio
W/cm.sup.3 cell(s) 1 1 13 9.2 1 -1 13 6.9 -1 1 7.2 9.2 -1 -1 7.2
6.9 0 0 10.1 8.28 0 0 10.1 8.28 0 0 10.1 8.28 0 0 10.1 8.28
[0036] The yield of extraction on a wet basis from fresh macroalga
is within a range of 2.9-6.6% (w/w); whereas the yield of
extraction for dry alga is within the range between 27.7 and 57.3%
(w/w); the yields for each of the algae species analyzed are
included in Table 2.
[0037] Maximum absorbance of this extract which is lyophilized and
subsequently redissolved in water, with respect to a water blank is
between 250 nm and 280 nm, as shown in FIG. 3.
Total Polyphenol Content:
[0038] The term polyphenols is understood to mean the entire family
of compounds present in plants, which contain or are derived from
the phenol group: benzoic acid derivatives, cinnamic acid
derivatives, flavonoids, etc.
[0039] A slight modification of the method proposed in Colorimetry
of total phenolics with phosphomolybdic phosphotungstic acid
reagents. Singleton & Rossi, Singleton, V. L.; Rossi, J. J. Am.
J. Enol. Vitic., 16, 144-158, 1965, was used to determine the
polyphenol content of the obtained extract. Thus, 500 .mu.l of
sample dissolved in water was used, to which 2.5 ml of
Folin-Ciocalteu reagent and 2.0 ml of Na.sub.2CO.sub.3 were added.
Absorbance was read at 765 nm, after incubating the samples for 15
min at 45.degree. C.
TABLE-US-00002 TABLE 2 Yields of aqueous extracts in dry and wet
basis, d.b.: considering 24 h .times. 45.degree. C. drying Species
Yield (%, w/w).sub.d.b. Yield (%, w/w).sub.w.b. Ascophyllum nodosum
38.0 6.6 Bifurcaria bifurcata 27.7 4.8 Saccorhiza polyschides 42.0
4.9 Sargassum muticum 57.3 2.9
[0040] The inhibition assay of the DPPH radical proposed in "Use of
a free radical method to evaluate antioxidant activity",
Brand-Williams W, Cuvelier M E, Berset C. LWT Food Sci Technol
28:5-30 18 (1995), was used to determine the proton-donating
capability of the extract. The method was applied with a slight
modification as described, in which 20 .mu.l of an aqueous solution
of the extract were added to 980 .mu.l of a methanolic solution of
the radical (6.9.times.10.sup.5 mol/l). The drop in absorbance was
recorded at 515 nm after 16 minutes. The percentage of inhibition
of the DPPH radical was calculated against the recording of a
blank. Concentration ranges of between 5 and 40 mg/ml of
Ascophyllum nodosum and Bifurcaria bifurcata extracts were
evaluated to determine EC50, the results of which were
23.73.+-.3.83 mg extract/ml and 17.68.+-.2.2 mg extract/ml,
respectively. These same values expressed as gallic acid
equivalents (GAE) correspond to 1.08.+-.0.16 and 1.04.+-.0.13 mg
GAE/ml, respectively.
TABLE-US-00003 TABLE 3 Characterization of lyophilized extracts
from four brown algae varieties. Total Uronic Total Trace elements
sugars Sulfates acid polyphenols Na K Mg P Ca S Species mg/g
extract Ascophyllum 115 50 47 44 70 46 9 2 6 18 nodosum Bifurcaria
156 118 55 62.4 20 71 3 1 4 36 bifurcata Saccorhiza 33 44 10 3 48
180 6 4 3 12 polyschides Sargassum 122 70 40 12 44 87 23 4 5 19
muticum
Polysaccharide Content
[0041] The term polysaccharides is understood to mean the polymers
formed from sugars, primarily hexoses and/or pentoses, which
function as a structural component and energy store in plants. The
polysaccharide content was determined by means of the
phenol-sulfuric acid method proposed in Colorimetric method for
determination of sugars and related substances, Dubois, M.; Gilles,
K. A.; Hamilton, J. K.; Rebers, P. A.; Smith, F. Anal. Chem., 28,
350-356, 1956. An average carbohydrate content of 156.7 mg of
glucose/g of lyophilizate of the extract obtained from the
macroalga Bifurcaria bifurcata was obtained, being 29% greater than
that obtained from Ascophyllum nodosum in similar conditions.
[0042] Fucoidans is understood to mean a family of polysaccharides
characteristic of algae, which are formed by polymerization of
derivatives of fucose, a sulfated sugar.
[0043] The average fucoidan content was determined by means of the
method described in Determination of inorganic sulphate in studies
on the enzymic and non-enzymic hydrolysis of carbohydrate and other
sulphate esters, Dodgson, Dodgson, K. S., Biochem. J., 78, pp.
312-317, 1961, and is expressed as total sulfates after digesting
the sample. The average value in the Bifurcaria bifurcata extracts
is 118 mg of sulfates/g of lyophilisate, 134% greater than an
Ascophyllum nodosum extract obtained in similar conditions. The
sulfur levels determined by means of ICP-OES techniques (mean of
36.3 mg S/g of lyophilisate) were twice those obtained for
Ascophyllum nodosum (mean of 18.14 mg/g).
[0044] The main polysaccharides in algae are the so-called
alginates, formed by polymerization primarily of glucuronic
acid.
[0045] The alginate content was estimated from the uronic acids
content according to the method proposed in New method for
quantitative determination of uronic acids, Blumenkrantz et al.,
Blumenkrantz, N. and Asboe-Hansen, G. Anal. Biochem, 54, pp.
484-489, 1973. The Bifurcaria bifurcata extract showed a mean
alginate content, expressed as uronic acids and using glucuronic
acid as a standard, of 55.1 mg/g of lyophilisate, being 15% greater
than an Ascophyllum nodosum extract obtained in the same
conditions.
Spectrophotometric Determination of Total Polyphenols
[0046] The Folin-Ciocalteu method for determination of total
polyphenols was used, using phloroglucinol as a standard. The
average total polyphenol content, which polyphenols were primarily
phlorotannins, in the Bifurcaria bifurcata extract, was of 62.4 mg
equivalents of phloroglucinol/g of lyophilizate, 7.3% greater than
that obtained from Ascophyllum nodosum.
Chromatographic Detection of Phlorotannins:
[0047] The term phlorotannins is understood to mean polymers
generated by polymerization of phloroglucinol
(1,3,5-trihydroxybenzene), a parallelism with the name tannins,
formed by polymerization of catechin/epicatechin and derivatives.
Tannins are cell wall components in plants and phlorotannins are
cell wall components in macroalgae.
[0048] Phlorotannins were extracted from the lyophilized extract,
according to the methodology proposed in High-performance liquid
chromatographic analysis of phlorotannins from the brown alga Fucus
vesiculosus Phytochem, Koivikko et al., 2007, Koivikko, R.,
Loponen, J., Pihlaja, K. and Jormalainen, V., Anal. 18, pp.
326-332, 2007, in which the steps of washing with hexane were
eliminated.
[0049] 95 mg of the Bifurcaria bifurcata extract were weighed and
solubilized in 25 ml of water; the phlorotannins were subsequently
extracted 4 times with 10 ml of an acetone/water: 7/3 mixture. The
sample was centrifuged to separate the supernatant. The supernatant
portions were pooled and acetone was removed with N.sub.2 stream.
The residue was resuspended in water and subsequently lyophilized.
The lyophilisate was dissolved in 1 ml of distilled water and was
ready to be injected into high-performance liquid chromatography
equipment. The chromatographic separation method was the one
proposed in Toxicity and antioxidant activity in vitro and in vivo
of two Fucus vesiculosus extracts, Zaragoza, M. C., Lopez, D.,
Saiz, M. P., Poquet, M., Perez, J., Puig-Parellada, P., Marmol, F.,
Simonetti, P., Gardana, C., Lerat, Y., Burtin, P., Inisan, C.,
Rousseau, I., Besnard, M., and Mitjavila, M. T. J. Agric. Food
Chem, 56, pp. 7773-7780, 2008.
[0050] FIG. 2 shows the major peaks (5-6, 11 and 18) the signal of
which is detected at 25-26, 30 and 37 min, respectively. Maximum
absorbance of the phlorotannin mixture is observed at 273 nm, as
shown in FIG. 3. The UV-Visible spectrum profile coincides with the
profile of a phloroglucinol molecule standard.
* * * * *